L-selectin is down-modulated from the cell surface in response to leukocyte activation or cross-linking with ligand mimetics such as anti-L-selectin antibodies or sulfatides. The down-modulation induced upon cellular activation with PMA is due to proteolytic shedding mediated by a cysteine metalloprotease, presumably the TNF-alpha converting enzyme (TALE), and is susceptible to inhibitors of the hydroxamate class. To determine if cross-linking-induced down-modulation of L-selectin is similarly affected by these inhibitors, we exposed Jurkat T cells to the anti-L-selectin antibody Dreg 200 or sulfatides in the presence of the hydroxamate TNF-alpha protease inhibitor (TAPI). TAPI completely inhibited PMA-induced downmodulation but had no effect on that induced by the anti-L-selectin antibody or sulfatides. The downmodulation induced by an anti-TCR antibody (WT31) was only partially inhibited by TAPI. An L-selectin mutant lacking the putative TACE cleavage site and resistant to PMA-induced shedding (321del.9) was expressed in L1.2 pre-B cells. Like WT L-selectin, this mutant was down-modulated when exposed to sulfatides, or Dreg 200 and this down-modulation was unaffected by TAPI. An L-selectin cytoplasmic tail deletion mutant (344del.15) expressed in L1.2 pre-B cells was down-modulated by PMA or sulfatides, but not Dreg 200. Electrophoretic analysis of L-selectin immunoprecipitated from the supernatants of Jurkat cells treated with either sulfatides or D200 revealed a proteolytic fragment of the same size as that released from the cell surface in response to PMA. Our data indicate that the down regulation of L-selectin in response to cross-linking by ligands or TCR engagement may be mechanistically distinct from that induced by PMA. Furthermore, our results with the 344del.15 mutant suggest that the down-modulation of Lselectin induced by certain sulfated carbohydrate ligands may be initiated through surface receptors other than L-selectin itself. The abbreviations used in this paper are: TAPI, TNF-alpha protease inhibitor; TACE, TNF-alpha converting enzyme; PMA, 4(3-phorbol 12-myristate 13-acetate.